1. THE PUZZLING PROPERTIES OF THE PERMEASE (PPP) Kim Finer, Jennifer Galovich, Ruth Gyure, Dave Westenberg March 4, 2006

2. IS THERE AN E.COLI-TYPE IRON PERMEASE GENE (FepD) IN CHROMOHALOBACTER SP. AND IF SO, IS THE PERIPLASMIC-FACING RESIDUE SEQUENCE SIGNIFICANTLY DIFFERENT?

3. BACKGROUND: Escherichia coli is known to have a ferric citrate transport system involving outer membrane permease, periplasmic transporters and an inner transmembrane protein encoded by the gene fepD. The fepD gene can also be found in other gram negative bacteria such as Pseudomonas aeruginosa PAO1. Chromohalobacter salexigens is a newly described species of bacterium able to survive in moderately saline environments. The species includes the previously described species, Halomonas elongata. One of its survival adaptations is the use of higher percentages of acidic amino acid residues in proteins that come into contact with the high salt environment. This modification protects the proteins from ‘salting out.’ HYPOTHESIS: We predict that transmembrane proteins such as FepD would also demonstrate this characteristic enrichment with acidic residues, but only in the hydrophilic regions facing the salty periplasm. Regions of protein hydrophilic regions facing the cytoplasm would be similar in charge makeup to the corresponding regions of FepD in E. coli, P. aeruginosa, etc.

4. http://hugroup.cems.umn.edu/Research/Images_research/Ecoli.jpg

5. SALTY ENVIRONMENT, For Chromobacter These segments would be the same in the three species These segments in Chromohalobacter would be more acidic than in the other two species SALTY ENVIRONMENT, For Chromohalobacter

6. METHODS We began by accessing an Excel spreadsheet of annotated C. salexigens sequences available at JGI. The spreadsheet was searched for ferric citrate transporter. The sequence reference was found (NP- 415122) and the homologous sequences from E. coli and P. aeruginosa were identified. The protein sequence for this accession was obtained from the JGI website. Protein sequences for FepD from E. coli K12 and P. aeruginosa (PAO1) were obtained from the NCBI website.

7. Using Biology Workbench, we uploaded these three aa sequences and used GREASE to reveal the hydrophobicity profile (graph) for each protein chain. Using PI, we obtained the overall isoelectric point at each pH, with our interest being pH7 to compare the three proteins overall. Our hypothesis predicts that Chromohalobacter would have the lowest isoelectric point (more acidic residues).

8. However, stronger evidence would be obtained by comparing each hydrophilic region in a pairwise fashion. TMAP allowed us to obtain a more precise prediction of which segments were hydrophobic vs. hydrophilic. There were 9 predicted transmembrane regions. Finally, each hydrophilic segment was submitted for PI analysis and the isoelectric point for each segment was compared among the three species. A third approach was to analyze the sequences using the Excel based analysis program, Protein Analysis. Protein analysis was used to plot the location of acidic amino acid residues along the entire peptide sequence.

9. E. coli Data

10. Sequence: MSGSVAVTRAIAVPGLLLLLIIATALSLLIGAKSLPASVV LEAFSGTCQS ADCTIVLDARLPRTLAGLLA GGALGLAGAL MQTLTRNPLA DPGLLGVNAGASFAIVLGAALFGYSSAQEQ LAMAFAGALV ASLIVAFTGS QGGGQLSPVRLTLAGVALAA VLEGLTSGIA LLNPDVYDQLRFWQAGSLDI RNLHTLKVVL IPVLIAGATA LLLSRALNSL SLGSDTATAL GSRVARTQLI GLLAITVLCG SATAIVGPIA FIGLMMPHMA RWLVGADHRW SLPVTLLATP ALLLFADIIG RVIVPGELRV SVVSAFIGAP VLIFLVRRKT RGGA Isoelectric Point: 10.476999

11. Kyte-Doolittle Hydropathy Profile

12. Predicted Transmembrane Segments for E. coli: TM 1: 11 - 39 (29) TM 2: 66 - 86 (21) TM 3: 94 - 114 (21) TM 4: 121 - 143 (23) TM 5: 152 - 173 (22) TM 6: 193 - 221 (29) TM 7: 243 - 271 (29) TM 8: 283 - 303 (21) TM 9: 310 - 330 (21)

13. pI of Non-Transmembrane Segments SegmentResiduespI 11 – 1011.045 240 – 656.265 387 – 933.099 4115 – 1203.294 5144 – 15111.045 6174 – 1924.171 7222 – 24210.888 8272 – 28210.888 9304 - 33412.406

14. P. aeruginosa Data

15. Sequence: MQASPMRRRRLRAWGLLAGALLLALAALASLALGSRPVPL AVTLDALQAV DPHDDRHLVV RELRLPRTLV ALLAGAALG VAGALMQALT RNPLAEPGLL GINAGAALAV IVGVALFDLA SMGQYLGCAF LGAGLAGIAV FLLGQARETG TNPVRLVLAG AGLSVMLASL TGIIVLNAPP EVFDRFRHWAAGSLSGSGFA LLGWPGLAIGAGLAAAFALAARLNALALGQEIGQALGVDLRL TWLLACLAVM LLAGAATALAGPIAFVGLVAP HLARLLAGPD QRWILPFSALIAAGLLLGADILGRLLAAPT EIAAGIVALL LGGPAFIVLVRRFRLSRL Isoelectric Point: 11.822999

16. Predicted Transmembrane Segments for P. aeruginosa: TM 1: 11 - 39 (29) TM 2: 66 - 94 (29) TM 3: 100 - 120 (21) TM 4: 126 - 146 (21) TM 5: 156 - 184 (29) TM 6: 202 - 226 (25) TM 7: 246 - 274 (29) TM 8: 289 - 309 (21) TM 9: 316 - 336 (21)

17. Kyte-Doolittle Hydropathy Profile

18. pI of Non-Transmembrane Segments SegmentResiduespI 1 1-1011.04 2 40-655.24 3 95-993.29 4 121-1255.92 5 147-1556.01 6 185-20111.04 7 227-2454.07 8 275-28811.04 9 310-3163.29

19. C. salexigens Data

20. Sequence: MLTRRTTRLA GLLAGLVLMA TTFAASVMLG TTELPPSTFI ATLLHYDPSR VAHIIIVKERLPRAVIAVLV GASLAIAGTL MQTLTRNPLA SPGILGINAG AMCFVVIAVA LLPLHAPADY VWAALLGALV AACLVLMLSR GGGRAGPSSL RVVLAGVAVT AMFVSFSQGL LIIDHQSFESVLYWLAGSVS GRELSLVVPL LPLFGIALLL CMLLVRHANA LMLGDDMVTS LGMHAGTIKL LLGLVVILLA GSSVALTGMI GFVGLIVPHM ARGLFGFDHR WLLPACALLG ACLLLLADVASRFLMPPQDV PVGVMTALIG TPFFIYLARR QQARP Isoelectric Point: 9.970999

21. Predicted Transmembrane Segments for C. salexigens: TM 1: 5 - 33 (29) TM 2: 62 - 90 (29) TM 3: 96 - 116 (21) TM 4: 122 - 142 (21) TM 5: 150 - 177 (28) TM 6: 194 - 222 (29) TM 7: 242 - 270 (29) TM 8: 281 - 308 (28)

22. Kyte-Doolittle Hydropathy Profile

23. pI of Non-Transmembrane Segments SegmentResiduespI 11 – 411.045 234 – 697.891 391 – 956.015 4117 – 1213.099 5143 – 14911.045 6178 – 1934.256 7223 – 2415.097 8271 – 28010.888 9309 – 33511.042

24. Comparison of pI Data SegmentE. coli P. aeruginosa C. salexigens 111.04511.0411.045 26.2655.247.891 33.0993.296.015 43.2945.923.099 511.0456.0111.045 64.17111.044.256 710.8884.075.097 810.88811.0410.888 912.4063.2911.042

25. Protein Analysis Program plot of acidic amino acids - E. coli

26. Protein Analysis Program plot of acidic amino acids - P. aeruginosa

27. Protein Analysis Program plot of acidic amino acids - C. salexigens

28. CONCLUSIONS Hydropathy plots illustrate a similar topology for the FepD proteins of C. salexigens, E. coli and P. aeruginosa. We predicted that the loop domains or the C. salexigens FepD protein, exposed to the exterior of the cell would have a lower pI than the exterior domains of E. coli and P. aeruginosa. The calculated pI of the entire C. salexigens FepD protein is lower than the pI of the other two organisms. However, the calculated pI of individual loop domains and a plot of acidic residues show that the overall hypothesis is not supported. Several loop domains of the C. salexigens FepD protein have a lower calculated pI than the corresponding segments of E. coli, However, comparison to the corresponding domains of the P. aeruginosa FepP protein do not show the same trend. In fact, P. aeruginosa seems to have more acidic loop domains than C. salexigens.